L-lysine-producing corynebacteria and process for the preparation of l-lysine

ABSTRACT

L-lysine-producing strains of  corynebacteria  with enhanced lysE gene (lysine export carrier gene), in which strains additional genes chosen from the group comprising the dapA gene (dihydrodipicolinate synthase gene), the lysC gene (aspartate kinase gene), the dapB gene (dihydrodipicolinate reductase gene) and the pyc gene, but especially the dapA gene and the lysC gene (aspartate kinase gene), are enhanced and, in particular, over-expressed, and to a process for the preparation of L-lysine.

This is a divisional of U.S. patent application Ser. No. 11/425,091,filed Jun. 19, 2006, now U.S. Pat. No. 7,435,584, issued on Oct. 14,2008, which is a divisional of U.S. patent application Ser. No.10/804,120, filed Mar. 19, 2004, now U.S. Pat. No. 7,094,584, issued onMar. 19, 2004, which is a continuation of U.S. patent application Ser.No. 10/337,985, filed Jan. 8, 2003, now U.S. Pat. No. 6,746,855, issuedon Jan. 8, 2003, which is a continuation of U.S. patent application Ser.No. 09/801,321, filed Mar. 8, 2001 (now abandoned), which is acontinuation-in-part of U.S. patent application Ser. No. 09/353,133,filed Jul. 14, 1999, now U.S. Pat. No. 6,200,785, issued on Mar. 13,1999, which claims priority to Germany Patent Appl. No. 199 31 317.2,filed Jul. 7, 1999.

The invention relates to L-lysine-producing strains of corynebacteriawith enhanced lysE gene (lysine export carrier gene), in which strainsadditional genes, chosen from the group comprising the dapA gene(dihydrodipicolinate synthase gene), the lysC gene (aspartate kinasegene), the dapB gene (dihydrodipicolinate reductase gene) and the pycgene, but especially the dapA gene and the lysC gene (aspartate kinasegene), are amplified and, in particular, over-expressed, and to aprocess for the preparation of L-lysine.

STATE OF THE ART

L-Lysine is a commercially important L-amino acid which is usedespecially as a feed additive in animal nutrition. The need has beensteadily increasing in recent years.

L-Lysine is prepared by a fermentation process with L-lysine-producingstrains of corynebacteria, especially Corynebacterium glutamicum.Because of the great importance of this product, attempts are constantlybeing made to improve the preparative process. Improvements to theprocess may relate to measures involving the fermentation technology,e.g. stirring and oxygen supply, or the composition of the nutrientmedia, e.g. the sugar concentration during fermentation, or the work-upto the product form, e.g. by ion exchange chromatography, or theintrinsic productivity characteristics of the microorganism itself.

The productivity characteristics of these microorganisms are improved byusing methods of mutagenesis, selection and mutant choice to givestrains which are resistant to antimetabolites, e.g.S-(2-aminoethyl)cysteine, or auxotrophic for amino acids, e.g.L-leucine, and produce L-lysine.

Methods of recombinant DNA technology have also been used for some yearsin order to improve L-lysine-producing strains of Corynebacteriumglutamicum by amplifying individual biosynthesis genes and studying theeffect on L-lysine production.

Thus, EP-A-0 088 166 reports the increase in productivity, afteramplification, of a DNA fragment conferring resistance toaminoethylcysteine. EP-B-0 387 527 reports the increase in productivity,after amplification, of an lysC allele coding for a feedback-resistantaspartate kinase. EP-B-0 197 335 reports the increase in productivity,after amplification, of the dapA gene coding for dihydrodipicolinatesynthase. EP-A-0 219 027 reports the increase in productivity, afteramplification, of the asd gene coding for aspartate semialdehydedehydrogenase. Pisabarro et al. (Journal of Bacteriology 175(9),2743-2749 (1993)) describe the dapB gene coding for dihydrodipicolinatereductase.

The effect of the amplification of primary metabolism genes on L-lysineproduction has also been studied. Thus EP-A-0 219 027 reports theincrease in productivity, after amplification, of the aspC gene codingfor aspartate aminotransferase. EP-B-0 143 195 and EP-B-0 358 940 reportthe increase in productivity, after amplification, of the ppc genecoding for phosphoenolpyruvate carboxylase. DE-A-198 31 609 reports theincrease in productivity, after amplification, of the pyc gene codingfor pyruvate carboxylase.

Finally, DE-A-195 48 222 describes that an increased activity of theL-lysine export carrier coded for by the lysE gene promotes lysineproduction.

In addition to these attempts to amplify an individual gene, attemptshave also been made to amplify two or more genes simultaneously andthereby to improve L-lysine production in corynebacteria. Thus, DE-A-3823 451 reports the increase in productivity, after simultaneousamplification, of the asd gene and the dapA gene from Escherichia coli.DE-A-39 43 117 discloses the increase in productivity, aftersimultaneous amplification, of an lysC allele coding for afeedback-resistant aspartate kinase and of the dapA gene by means ofplasmid pJC50. EP-A-0 841 395 particularly reports the increase inproductivity, after simultaneous amplification, of an lysC allele codingfor a feedback-resistant aspartate kinase and of the dapB gene; furtherimprovements could be achieved by additional amplification of the dapB,lysA and ddh genes. EP-A-0 854 189 describes the increase inproductivity, after simultaneous amplification, of an lysC allele codingfor a feedback-resistant aspartate kinase and of the dapA, dapB, lysAand aspC genes. EP-A-0 857 784 particularly reports the increase inproductivity, after simultaneous amplification, of an lysC allele codingfor a feedback-resistant aspartate kinase enzym and of the lysA gene; afurther improvement could be achieved by additional amplification of theppc gene.

It is clear from the many processes described in the state of the artthat there is a need for the development of novel approaches and for theimprovement of existing processes for lysine production withcorynebacteria.

OBJECT OF THE INVENTION

The object of the invention consists in using novel measures to provideimproved L-lysine-producing strains of corynebacteria.

DETAILED DESCRIPTION OF THE INVENTION

L-Lysine is a commercially important L-amino acid which is usedespecially as a feed additive in animal nutrition.

When L-lysine or lysine is mentioned in the following text, it isunderstood as meaning not only the base but also the appropriate salts,e.g. lysine hydrochloride or lysine sulfate.

The invention provides L-lysine-producing strains of corynebacteriaenhanced lysE gene (lysine export carrier gene), wherein theyadditionally contain genes chosen from the group comprising the dapAgene (dihydrodipicolinate synthase gene), the lysC gene (aspartatekinase gene), the dapB gene (dihydrodipicolinate reductase gene) and thepyc gene (pyruvate carboxylase gene), but especially the dapA gene andthe lysC gene, which, individually or together, are enhanced and,preferably, over-expressed.

The novel DNA sequence located upstream (5′ end) from the dapB gene hasalso been found which carries the −35 region of the dapB promoter and isadvantageous for the expression of the dapB gene. It is shown as SEQ IDNo. 1.

A corresponding DNA capable of replication, with the nucleotide sequenceshown in SEQ ID No. 1, is therefore claimed as well.

The invention also provides the MC20 or MA16 mutations of the dapApromoter shown in SEQ ID No. 5 and SEQ ID No. 6, deposited underDSM12868 and DSM12867 respectively.

The invention also provides L-lysine-producing strains of corynebacteriawith enhanced lysE gene, wherein additionally the dapA and dapB genesare simultaneously enhanced and, in particular, over-expressed.

Finally, the invention also provides L-lysine-producing strains ofcorynebacteria with enhanced lysE gene, wherein additionally the dapAand lysC genes are simultaneously enhanced and, in particular,over-expressed.

In this context the term “enhancement” describes the increase in theintracellular activity, in a microorganism, of one or more enzymes whichare coded for by the appropriate DNA, by increasing the copy number ofthe gene(s), using a strong promoter or using a gene coding for anappropriate enzyme with a high activity, and optionally combining thesemeasures.

In this context, “amplification” describes a specific procedure forachieving an enhancement whereby the number of DNA molecules carrying agene or genes, an allele or alleles, a regulatory signal or signals orany other genetic feature(s) is increased.

A process for the preparation of L-lysine by the fermentation of thesecorynebacteria is also claimed.

The microorganisms which the present invention provides can prepareL-lysine from glucose, sucrose, lactose, fructose, maltose, molasses,starch or cellulose or from glycerol and ethanol, especially fromglucose or sucrose. Said microorganisms are corynebacteria, especiallyof the genus Corynebacterium. The species Corynebacterium glutamicum maybe mentioned in particular in the genus Corynebacterium, being known tothose skilled in the art for its ability to produce amino acids. Thisspecies includes wild-type strains such as Corynebacterium glutamicumATCC13032, Brevibacterium flavum ATCC14067, Corynebacterium melassecolaATCC17965 and strains or mutants derived therefrom. Examples ofL-lysine-producing mutants of corynebacteria are:

-   -   Corynebacterium glutamicum FERM-P 1709    -   Brevibacterium flavum FERM-P 1708    -   Brevibacterium lactofermentum FERM-P 1712    -   Brevibacterium flavum FERM-P 6463    -   Brevibacterium flavum FERM-P 6464    -   Corynebacterium glutamicum DSM5714    -   Corynebacterium glutamicum DSM12866

DE-A-195 48 222 discloses the advantageous effect of over-expression ofthe lysE gene on L-lysine production.

The additional enhanced expression of the dapB gene or the pyc gene, orin particular an additionally enhanced expression of an lysC allelecoding for a feedback-resistant aspartate kinase, or in particular anadditionally enhanced expression of the dapA gene, improves L-lysineproduction.

The inventors have also found that, for a given over-expression of thelysE gene, the simultaneous, additionally enhanced expression of thedapA and dapB genes brings further advantages for L-lysine production.

A corresponding DNA capable of replication, with the nucleotide sequenceshown in SEQ ID No. 1, is therefore claimed as well.

For a given over-expression of the lysE gene, the simultaneous,additionally enhanced expression of the dapA gene and the lysC allele isalso advantageous.

An enhancement (over-expression) is achieved e.g. by increasing the copynumber of the appropriate genes or mutating the promoter and regulatoryregion or the ribosome binding site located upstream from the structuralgene. Expression cassettes incorporated upstream from the structuralgene work in the same way. Inducible promoters additionally make itpossible to increase the expression in the course of the formation ofL-lysine by fermentation. Measures for prolonging the life of the m-RNAalso improve the expression. Furthermore, the enzyme activity is alsoenhanced by preventing the degradation of the enzyme protein, the genesor gene constructs either being located in plasmids (shuttle vectors) ofvariable copy number or being integrated and amplified in thechromosome. Alternatively, it is also possible to achieve anover-expression of the genes in question by changing the composition ofthe media and the culture technique.

Those skilled in the art will find relevant instructions inter alia inMartin et al. (Bio/Technology 5, 137-146 (1987)), Guerrero et al. (Gene138, 35-41 (1994)), Tsuchiya and Morinaga (Bio/Technology 6, 428-430(1988)), Eikmanns et al. (Gene 102, 93-98 (1991)), EP-0 472 869, U.S.Pat. No. 4,601,893, Schwarzer and Pühler (Bio/Technology 9, 84-87(1991)), Reinscheid et al. (Applied and Environmental Microbiology 60,126-132 (1994)), LaBarre et al. (Journal of Bacteriology 175, 1001-1007(1993)), WO 96/15246, Malumbres et al. (Gene 134, 15-24 (1993)),JP-A-10-229891, Jensen and Hammer (Biotechnology and Bioengineering 58,191-195 (1998)) or the handbook “Manual of Methods for GeneralBacteriology” of the American Society for Bacteriology (Washington D.C.,USA, 1981) and well-known textbooks on genetics and molecular biology.

The genes from Corynebacterium glutamicum used according to theinvention are described and can be isolated, prepared or synthesized byknown methods.

Methods of localized mutagenesis are described inter alia by Higuchi etal. (Nucleic Acids Research 16: 7351-7367 (1988)) or by Silver et al. inthe handbook by Innis, Glefand and Sninsky (eds.) entitled PCRStrategies (Academic Press, London, UK, 1995).

The first step in isolating a gene of interest from C. glutamicum is toconstruct a gene library of this microorganism in e.g. E. coli oroptionally also in C. glutamicum. The construction of gene libraries isdocumented in generally well-known textbooks and handbooks. Exampleswhich may be mentioned are the textbook by Winnacker entitled From Genesto Clones, Introduction to Gene Technology (Verlag Chemie, Weinheim,Germany, 1990) or the handbook by Sambrook et al. entitled MolecularCloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press,1989). Bathe et al. (Molecular and General Genetics 252: 255-265 (1996))describe a gene library of C. glutamicum ATCC13032 which was constructedusing cosmid vector SuperCos I (Wahl et al., Proceedings of the NationalAcademy of Sciences USA, 84: 2160-2164 (1987)) in E. coli K-12 NM554(Raleigh et al., Nucleic Acids Research 16: 1563-1575 (1988)). Bormannet al. (Molecular Microbiology 6(3), 317-326) in turn describe a genelibrary of C. glutamicum ATCC13032 using cosmid pHC79 (Hohn and Collins,Gene 11, 291-298 (1980)).

A gene library of C. glutamicum in E. coli can also be constructed usingplasmids like pBR322 (Bolivar, Life Sciences 25, 807-818 (1979)) orpUC19 (Norrander et al., Gene, 26: 101-106 (1983)). In the same way itis also possible to use shuttle vectors such as pJC1 (Cremer et al.,Molecular and General Genetics 220, 478-480 (1990)) or pEC5 (Eikmanns etal., Gene 102, 93-98 (1991)), which replicate in E. coli and C.glutamicum. Restriction- and/or recombination-defective strains areparticularly suitable hosts, an example being the E. coli strainDH5αmcr, which has been described by Grant et al. (Proceedings of theNational Academy of Sciences, USA 87, 4645-4649 (1990)). Other examplesare the restriction-defective C. glutamicum strains RM3 and RM4, whichare described by Schafer et al. (Applied and Environmental Microbiology60(2), 756-759 (1994)).

The gene library is then transferred to an indicator strain bytransformation (Hanahan, Journal of Molecular Biology 166, 557-580(1983)) or electroporation (Tauch et al., FEMS Microbiological Letters,123: 343-347 (1994)). The characteristic feature of the indicator strainis that it possesses a mutation in the gene of interest which causes adetectable phenotype, e.g. an auxotrophy. The indicator strains ormutants are obtainable from publicized sources or strain collections,e.g. the Genetic Stock Center of Yale University (New Haven, Conn.,USA), or if necessary are specially prepared. An example of such anindicator strain which may be mentioned is the E. coli strain RDA8requiring mesodiaminopimelic acid (Richaud et al., C.R. Acad. Sci. ParisSer. III 293: 507-512 (1981)), which carries a mutation (dapA::Mu) inthe dapA gene.

After transformation of the indicator strain with a recombinant plasmidcarrying the gene of interest, and expression of the gene in question,the indicator strain becomes prototrophic in respect of the appropriatecharacteristic. If the cloned DNA fragment confers resistance, e.g. toan antimetabolite like S-(2-aminoethyl)cysteine, the indicator straincarrying the recombinant plasmid can be identified by selection onappropriately supplemented nutrient media.

If the nucleotide sequence of the gene region of interest is known orobtainable from a data bank, the chromosomal DNA can be isolated byknown methods, e.g. as described by Eikmanns et al. (Microbiology 140,1817-1828 (1994)), and the gene in question can be synthesized by thepolymerase chain reaction (PCR) using suitable primers and cloned into asuitable plasmid vector, e.g. pCRIITOPO from Invitrogen (Groningen, TheNetherlands). A summary of PCR methodology can be found in the book byNewton and Graham entitled PCR (Spektrum Akademischer Verlag,Heidelberg, Germany, 1994).

Examples of publicly accessible data banks for nucleotide sequences arethat of the European Molecular Biologies Laboratories (EMBL, Heidelberg,Germany) or that of the National Center for Biotechnology Information(NCBI, Bethesda, Md., USA).

The isolation and cloning of the lysE gene from C. glutamicum ATCC13032,together with the nucleotide sequence, are described in DE-A-195 48 222.

The isolation, cloning and sequencing of the dapA gene from variousstrains of C. glutamicum are described by Cremer et al. (Molecular andGeneral Genetics 220: 478-480 (1990)), by Pisabarro et al. (Journal ofBacteriology 175: 2743-2749 (1993)) and by Bonnassie et al. (NucleicAcids Research 18, 6421 (1990)). The nucleotide sequence of the dapAgene is obtainable under accession number X53993.

The isolation, cloning and sequencing of the dapB gene fromBrevibacterium lactofermentum are described by Pisabarro et al. (Journalof Bacteriology 175: 2743-2749 (1993)). The nucleotide sequence of thedapB gene is obtainable under accession number X67737.

The isolation, cloning and sequencing of the lysC gene and of lysCalleles coding for a feedback-resistant aspartate kinase are reported byseveral authors. Thus, Kalinowski et al. (Molecular and General Genetics224: 317-324 (1990)) report the lysC allele from the C. glutamicumstrain DM58-1. DE-A-39 43 117 reports the cloning of the lysC allelefrom the C. glutamicum strain MH20. Follettie et al. (Journal ofBacteriology 175: 4096-4103 (1993)) report the lysC allele from the C.flavum strain N13, which is called ask in said publication. Kalinowskiet al. (Molecular Microbiology 5, 1197-1204 (1991)) report the lysC genefrom C. glutamicum ATCC13032. The nucleotide sequences of the lysC geneand of various lysC alleles are obtainable inter alia under accessionnumbers X57226 and E06826.

The genes obtained in this way can then be incorporated inter alia intoplasmid vectors, e.g. pJC1 (Cremer et al., Molecular and GeneralGenetics 220, 478-480 (1990)) or pEC5 (Eikmanns et al., Gene 102, 93-98(1991)), individually or in suitable combinations, transferred todesired strains of corynebacteria, e.g. the strain MH20-22B (Schrumpf etal., Applied Microbiology and Biotechnology 37: 566-571 (1992)), bytransformation, e.g. as in Thierbach et al. (Applied Microbiology andBiotechnology 29, 356-362 (1988)), or by electroporation, e.g. as inDunican and Shivnan (Bio/Technology 7, 1067-1070 (1989)), and expressed.The strain to be chosen can equally well be transformed with two plasmidvectors, each containing the gene or genes in question, therebyachieving the advantageous, simultaneously enhanced expression of two ormore genes in addition to the known enhancement of the lysE gene.

Examples of such strains are:

-   -   the strain MH20-22B/pJC33/pEC7lysE, in which the lysE and lysC        genes are expressed with simultaneous enhancement, or    -   the strain MH20-22B/pJC50/pEC7lysE, in which the lysE, lysC and        dapA genes are expressed with simultaneous enhancement, or    -   the strain MH20-22B/pJC23/pEC7lysE, in which the lysE and dapA        genes are expressed with simultaneous enhancement, or    -   the strain MH20-22B/pJC23/pEC7dapBlysE, in which the lysE, dapA        and dapB genes are expressed with simultaneous enhancement.

The microorganisms prepared according to the invention can be cultivatedfor L-lysine production continuously or discontinuously by the batchprocess, the fed batch process or the repeated fed batch process. Asummary of known cultivation methods is provided in the textbook byChmiel (Bioprozesstechnik 1. Einführung in die Bioverfahrenstechnik(Bioprocess Technology 1. Introduction to Bioengineering) (GustavFischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas(Bioreaktoren and periphere Einrichtungen (Bioreactors and PeripheralEquipment) (Vieweg Verlag, Brunswick/Wiesbaden, 1994)).

The culture medium to be used must appropriately meet the demands of theparticular microorganisms. Descriptions of culture media for variousmicroorganisms can be found in the handbook “Manual of Methods forGeneral Bacteriology” of the American Society for Bacteriology(Washington D.C., USA, 1981).

Carbon sources which can be used are sugars and carbohydrates, e.g.glucose, sucrose, lactose, fructose, maltose, molasses, starch andcellulose, oils and fats, e.g. soya oil, sunflower oil, groundnut oiland coconut fat, fatty acids, e.g. palmitic acid, stearic acid andlinoleic acid, alcohols, e.g. glycerol and ethanol, and organic acids,e.g. acetic acid. These substances can be used individually or as amixture.

Nitrogen sources which can be used are organic nitrogen-containingcompounds such as peptones, yeast extract, meat extract, malt extract,corn steep liquor, soybean flour and urea, or inorganic compounds suchas ammonium sulfate, ammonium chloride, ammonium phosphate, ammoniumcarbonate and ammonium nitrate. The nitrogen sources can be usedindividually or as a mixture.

Phosphorus sources which can be used are potassium dihydrogenphosphateor dipotassium hydrogenphosphate or the corresponding sodium salts. Theculture medium must also contain metal salts, e.g. magnesium sulfate oriron sulfate, which are necessary for growth. Finally, essentialgrowth-promoting substances such as amino acids and vitamins can be usedin addition to the substances mentioned above. Said feed materials canbe added to the culture all at once or fed in appropriately duringcultivation.

The pH of the culture is controlled by the appropriate use of basiccompounds such as sodium hydroxide, potassium hydroxide or ammonia, oracid compounds such as phosphoric acid or sulfuric acid. Foaming can becontrolled using antifoams such as fatty acid polyglycol esters. Thestability of plasmids can be maintained by optionally adding suitableselectively acting substances, e.g. antibiotics, to the medium. Aerobicconditions are maintained by introducing oxygen or oxygen-containinggaseous mixtures, e.g. air, into the culture. The temperature of theculture is normally 20° C. to 45° C. and preferably 25° C. to 40° C. Theculture is continued until L-lysine formation has reached a maximum.This objective is normally achieved within 10 hours to 160 hours.

The concentration of L-lysine formed can be determined with the aid ofamino acid analyzers by means of ion exchange chromatography andpostcolumn reaction with ninhydrin detection, as described by Spackmannet al. (Analytical Chemistry 30, 1190 (1958)).

The following microorganisms have been deposited in the DeutscheSammlung für Mikroorganismen and Zellkulturen (German Collection ofMicroorganisms and Cell Cultures (DSMZ), Brunswick, Germany) under theterms of the Budapest Treaty:

-   -   Escherichia coli K-12 strain DH5α/pEC7lysE as DSM12871    -   Escherichia coli K-12 strain DH5α/pEC7dapBlysE as DSM12875    -   Corynebacterium glutamicum strain DSM5715/pJC23 as DSM12869    -   Corynebacterium glutamicum strain DSM5715aecD::dapA(MA16) as        DSM12867    -   Corynebacterium glutamicum strain DSM5715aecD::dapA(MC20) as        DSM12868

BRIEF DESCRIPTION OF THE DRAWINGS

The following Figures are attached:

FIG. 1: Plasmid pEC7lysE

FIG. 2: Plasmid pEC7dapB

FIG. 3: Plasmid pEC7dapBlysE

The abbreviations used in the Figures are defined as follows:

-   Cm: chloramphenicol resistance gene-   dapB: dapB gene from C. glutamicum-   lysE: lysE gene from C. glutamicum-   pyc: pyc gene from C. glutamicum-   OriE: plasmid-coded origin of replication of E. coli-   pBL: DNA fragment of plasmid pBL1-   EcoRI: cleavage site of the restriction enzyme EcoRI-   EcoRV: cleavage site of the restriction enzyme EcoRV-   HincII: cleavage site of the restriction enzyme HincII-   HindIII: cleavage site of the restriction enzyme HindIII-   KpnI: cleavage site of the restriction enzyme-   KpnISalI: cleavage site of the restriction enzyme-   SalISmaI: cleavage site of the restriction enzyme-   SmaISphI: cleavage site of the restriction enzyme SphI-   PvuII: cleavage site of the restriction enzyme PvuII-   BamHI: cleavage site of the restriction enzyme BamHI

Example 1 Preparation of the DNA Coding for lysE

Chromosomal DNA was isolated from the strain ATCC13032 by theconventional methods (Eikmanns et al., Microbiology 140: 1817-1828(1994)). The polymerase chain reaction (PCR) was used to amplify a DNAfragment carrying the lysE gene. The following primer oligonucleotideswere chosen for the PCR on the basis of the lysE gene sequence known forC. glutamicum (Vrljic et al., Molecular Microbiology 22(5), 815-826(1996)) (accession number X96471):

LysBam1: (SEQ ID NO: 7) 5′ CTC GAG AGC (GGA TCC) GCG CTG ACT CAC C 3′LysBam2: (SEQ ID NO: 8) 5′ GGA GAG TAC GGC (GGA TCC) ACC GTG ACC 3′

The primers shown were synthesized by MWG Biotech (Ebersberg, Germany)and the PCR was carried out by the standard PCR method of Innis et al.(PCR Protocols. A Guide to Methods and Applications, 1990, AcademicPress). The primers make it possible to amplify an approx. 1.1 kb DNAfragment carrying the lysE gene. The primers also contain the sequencefor the cleavage site of the restriction endonuclease BamHI, which isindicated by brackets in the nucleotide sequence shown above.

The amplified DNA fragment of approx. 1.1 kb, carrying the lysE gene,was identified by means of electrophoresis in 0.8% agarose gel, isolatedfrom the gel and purified with the QIAquick Gel Extraction Kit (cat. no.28704) from Qiagen (Hilden, Germany). The fragment was then ligated bymeans of T4 DNA ligase from Boehringer Mannheim (Mannheim, Germany) tovector pUC18 (Norrander et al., Gene (26) 101-106 (1983)). This was doneby fully cleaving vector pUC18 with the restriction endonuclease SmaIand treating it with alkaline phosphatase (Boehringer Mannheim,Mannheim, Germany). The ligation mixture was transformed to the E. colistrain DH5α(Hanahan, in: DNA Cloning, A Practical Approach, Vol. I,IRL-Press, Oxford, Washington D.C., USA). Plasmid-carrying cells wereselected by plating the transformation mixture on LB agar (Sambrook etal., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.) which had been supplementedwith 50 mg/l of ampicillin. Plasmid DNA was isolated from a transformantand checked by treatment with the restriction enzyme BamHI followed byagarose gel electrophoresis. The plasmid was called pUC18lysE.

Example 2 Preparation of dapB

Chromosomal DNA was isolated from the Corynebacterium glutamicum strainATCC13032 as indicated in Example 1. The sequence of the dapB gene assuch from Corynebacterium glutamicum is known (accession number X67737).However, the published DNA sequence comprises only 56 by upstream fromthe translation start, so the 5′ end upstream from the translation startwas additionally sequenced.

The sequencing was carried out with plasmid pJC25 (EP-B 0 435 132) usinga primer oligonucleotide which binds in the region of the known dapBsequence (accession number X67737). The sequence of the sequencingprimer used was:

5′ GAA CGC CAA CCT TGA TTC C 3′ (SEQ ID NO: 9)

The sequencing was carried out by the chain termination method describedby Sanger et al., Proc. Natl. Acad. Sci. USA, (74), 5463-5467 (1977).The sequencing reaction was performed with the aid of the AutoReadSequencing Kit (Pharmacia, Freiburg). The electrophoretic analysis anddetection of the sequencing products were carried out with the A.L.F.DNA sequencer from Pharmacia (Freiburg, Germany).

The DNA sequence obtained was used to choose a second primer in order toobtain further sequence data upstream from the transcription start. Thefollowing primer was chosen for this purpose:

5′ CTT TGC CGC CGT TGG GTT C 3′ (SEQ ID NO: 10)

The sequencing reaction was carried out as described above. The novelsequence upstream from the dapB gene is shown as SEQ ID No. 1. Thesequence including the nucleotide sequence of the dapB gene is shown asSEQ ID No. 2.

The polymerase chain reaction was used to amplify the dapB gene. Forthis purpose, two primer oligonucleotides, chosen on the basis of theknown DNA sequence of the dapB gene, were synthesized by MWG Biotech:

P-dap: (SEQ ID NO: 11) 5′ (AAG CTT) AGG TTG TAG GCG TTG AGC 3′ dapa11:(SEQ ID NO: 12) 5′ TTA ACT TGT TCG GCC ACA GC 3′

The 5′ primer (primer P-dap) contains a HindIII cleavage site which isindicated by brackets in the sequence shown above. The PCR was carriedout as in Example 1. An approx. 1.1 kb DNA fragment, which carries thedapB gene and contains a cleavage site for the restriction endonucleaseHindIII at one end, was amplified in this way. The PCR fragment obtainedwas purified from 0.8% agarose gel (QIAquick Gel Extraction Kit fromQiagen, Hilden, Germany) and cloned into cloning vector pCR2.1TOPO(Invitrogen, Leek, The Netherlands) with the TOPO TA Cloning Kit(Invitrogen, Leek, The Netherlands, cat. no. K4550-01). The ligationmixture was transformed to the E. coli strain TOP10F′ from Invitrogen,the transformation mixture was plated on LB agar containing kanamycin(50 mg/l), IPTG (0.16 mM) and X-Gal (64 mg/l) and kanamycin-resistant,white colonies were isolated. Plasmid DNA was isolated from atransformant with the aid of the QIAprep Spin Miniprep Kit from Qiagenand checked by cleavage with the restriction enzyme HindIII followed byagarose gel electrophoresis. The DNA sequence of the amplified DNAfragment was checked by sequencing. The sequence of the PCR productmatches the sequence shown in SEQ ID No. 1. The plasmid obtained wascalled pCR2.1TOPOdapB.

Example 3 Cloning of lysE into Vector pEC7

The lysE-carrying fragment from plasmid pUC18lysE (Example 1) wasinserted into vector pEC7 as described below. Vector pEC7 is based on E.coli —C. glutamicum shuttle vector pEC5 (Eikmanns et al., Gene 102:93-98 (1991)). The BamHI cleavage site not located in the polylinker wasremoved from plasmid pEC5 in the following manner: Plasmid pEC5 waspartially cleaved with the restriction enzyme BamHI. The approx. 7.2 kbDNA fragment was isolated from the agarose gel and the protruding endswere filled in with Klenow polymerase (Boehringer Mannheim). Theresulting DNA fragment was ligated (T4 ligase, Boehringer Mannheim). Theligation mixture was transformed to the E. coli strain DH5α andchloramphenicol-resistant colonies were isolated on LB agar containingchloramphenicol (50 mg/l). Plasmid DNA was isolated from a transformant(QIAprep Spin Miniprep Kit from Qiagen) and checked by restrictioncleavage with the restriction enzymes BamHI and PstI. The resultingplasmid was called pEC6.

Plasmid pEC6 was fully cleaved with the restriction enzyme XhoI. A DNAfragment carrying the trp terminator was ligated to vector DNA fragment(T4 ligase, Boehringer Mannheim). The ligation mixture was transformedto the E. coli strain DH5α and kanamycin-resistant colonies wereisolated on LB agar containing kanamycin (50 mg/l). Plasmid DNA wasisolated from a transformant (QIAprep Spin Miniprep Kit from Qiagen) andchecked by restriction cleavage with the restriction enzymes BamHI andXhoI. The resulting plasmid was called pEC7.

Plasmid pUC18lysE described in Example 1 was fully digested with therestriction enzyme BamHI and the 1.1 kb BamHI fragment carrying the lysEgene was isolated as in Example 1. Vector pEC7 was likewise fullycleaved with the restriction enzyme BamHI and treated with alkalinephosphatase. The BamHI vector fragment and the BamHI lysE fragment wereligated (Rapid DNA Ligation Kit, Boehringer Mannheim) and transformed tothe E. coli strain DH5α. Plasmid-carrying transformants were selected onLB agar containing chloramphenicol (10 mg/l). Plasmid DNA was isolated(QIAprep Spin Miniprep Kit, Qiagen) and checked by restriction cleavagewith the enzyme BamHI. The resulting plasmid was called pEC7lysE (FIG.1). The strain obtained by transformation of plasmid pEC7lysE to the E.coli strain DH5α was called DH5α/pEC7lysE.

Example 4 Cloning of dapB into Vector pEC7

An approx. 1.1 kb DNA fragment carrying the dapB gene was isolated fromplasmid pCR2.1TOPOdapB (from Example 2). For this purpose, plasmidpCR2.1TOPOdapB was fully digested with the restriction enzyme HindIIIand the approx. 1.1 kb DNA fragment carrying the dapB gene was isolated.

The dapB-carrying DNA fragment obtained was ligated to vector pEC7(Example 3) (T4 DNA ligase, Boehringer Mannheim), which had also beenfully digested with the restriction enzyme HindIII and treated withalkaline phosphatase (Boehringer Mannheim). The ligation mixture wastransformed to the E. coli strain DH5α and kanamycin-resistant colonieswere isolated on LB agar containing kanamycin (50 mg/l). Plasmid DNA wasisolated from a transformant (QIAprep Spin Miniprep Kit from Qiagen) andchecked by restriction cleavage with the restriction enzyme HindIII. Theresulting plasmid was called pEC7dapB (FIG. 2). The Escherichia colistrain obtained was called DH5α/pEC7dapB.

Example 5 Preparation of a Plasmid Simultaneously Containing dapB andlysE

The dapB gene was isolated as a HindIII fragment from plasmidpCR2.1TOPOdapB containing the dapB gene from C. glutamicum ATCC13032. Todo this, the plasmid was fully digested with the restriction enzymeHindIII and the dapB-carrying DNA fragment was isolated from 0.8%agarose gel (QIAquick Gel Extraction Kit, Qiagen). Vector pEC7 (Example3) was also fully digested with the restriction enzyme HindIII andtreated with alkaline phosphatase. The 1.1 kb fragment containing dapBwas ligated to the resulting linear vector fragment (T4 ligase,Boehringer Mannheim) and the ligation mixture was transformed to the E.coli strain DH5α. Plasmid-carrying transformants were selected on LBagar containing chloramphenicol (10 mg/l). Plasmid DNA was isolated(QIAprep Spin Miniprep Kit, Qiagen, Hilden, Germany) and checked byrestriction cleavage with the restriction enzyme HindIII.

The resulting plasmid was called pEC7lysEdapB. This plasmid is capableof autonomous replication in Escherichia coli and in Corynebacterium andconfers resistance to the antibiotic chloramphenicol on its host.

As shown in FIG. 3, plasmid pEC7lysEdapB simultaneously contains thedapB gene, which codes for dihydrodipicolinate reductase, and the lysEgene, which codes for the lysine exporter. The strain obtained bytransformation of the E. coli strain DH5α with pEC7lysEdapB was calledDH5α/pEC7lysEdapB.

Example 6 Transformation of the Strain MH20-22B with Plasmids pJC1,pJC33 and pJC50

Plasmid pJC1 is a plasmid capable of replication in Escherichia coli andCorynebacterium glutamicum (Cremer et al., Molecular and GeneralGenetics 220: 478-480 (1990)). Plasmid pJC33 (Cremer et al., Applied andEnvironmental Microbiology 57(6), 1746-1752 (1991)), which carries thelysC(Fbr) gene from the C. glutamicum strain MH20-22B, is derivedtherefrom.

Plasmid pJC50 is also based on vector pJC1 and carries the lysC(FBR)gene from C. glutamicum MH20-22B and the dapA gene from C. glutamicumATCC13032 (DE-A-39 43 117).

Plasmids pJC1, pJC33 and pJC50 were introduced into the strain MH20-22Bby the electroporation method (Haynes and Britz, FEMS MicrobiologyLetters (61) 329-334 (1989)). The C. glutamicum strain MH20-22B is anAEC-resistant lysine producer deposited under the number DSM5715.

The transformants obtained by means of electroporation were isolated onselection agar (LBHIS agar (18.5 g/l of brain-heart infusion broth, 0.5M sorbitol, 5 g/l of bacto tryptone, 2.5 g/l of bacto yeast extract, 5g/l of NaCl, 18 g/l of bacto agar)) containing 15 mg/l of kanamycin.Plasmid DNA was isolated by the conventional methods (Peters-Wendisch etal., Microbiology 144, 915-927 (1998)), cleaved with suitablerestriction endonucleases and checked. The strains obtained were calledMH20-22B/pJC1, MH20-22B/pJC33 and MH20-22B/pJC50.

Example 7 Transformation with Plasmids pEC7lysE and pEC7dapBlysE

The strains prepared in Example 6 were subsequently provided with asecond plasmid.

Plasmids pEC7lysE and pEC7dapBlysE were introduced by theelectroporation method into the strains MH20-22B/pJC1, MH20-22B/pJC33and MH20-22B/pJC50 described.

The transformed bacteria are selected on the basis of the antibioticresistance of the plasmids they contain. The transformants obtained bymeans of electroporation were isolated on selection agar (LBHIS agarcontaining 15 mg/l of kanamycin and 7.5 mg/l of chloramphenicol).Plasmid DNA was isolated, cleaved with suitable restrictionendonucleases and checked.

Example 8 Preparation of Lysine

The various C. glutamicum strains obtained in Example 7 were cultivatedin a nutrient medium suitable for lysine production and the lysinecontent of the culture supernatant was determined.

This was done by first incubating the various strains on agar plateswith the appropriate antibiotics (brain-heart agar containing kanamycin(25 mg/l), chloramphenicol (10 mg/l)) for 24 hours at 33° C. These agarplate cultures were used to inoculate a preculture (10 ml of medium in a100 ml conical flask). Complete medium CgIII was used as the preculturemedium. Kanamycin (25 mg/l) and chloramphenicol (10 mg/l) were added.The preculture was incubated for 24 hours at 33° C. on a shaker at 240rpm. This preculture was used to inoculate a main culture to give aninitial OD (660 nm) of 0.2 OD. Medium MM was used for the main culture.

Medium MM CSL (corn steep liquor) 5 g/l MOPS (morpholinopropane 20 g/lsulfonic acid) Glucose 50 g/l (autoclave separately) Salts: (NH₄)₂SO₄ 25g/l KH₂PO₄ 0.1 g/l MgSO₄*7H₂O 1.0 g/l CaCl₂*2H₂O 10 mg/l FeSO₄*7H₂O 10mg/l MnSO₄*H₂O 5.0 mg/l Biotin 0.3 mg/l (sterile-filtered) Thiamine*HCl0.2 mg/l (sterile-filtered) CaCO₃ 25 g/l

CSL, MOPS and the salt solution are adjusted to pH 7 with aqueousammonia and autoclaved. The sterile substrate and vitamin solutions andthe dry-autoclaved CaCO₃ are then added.

Cultivation is carried out in a volume of 10 ml in a 100 ml conicalflask with baffles. Kanamycin (25 mg/l) and chloramphenicol (10 mg/l)were added. Cultivation proceeded at 33° C. and 80% atmospherichumidity.

After 48 hours the OD was measured at a wavelength of 660 nm with aBiomek 1000 (Beckmann Instruments GmbH, Munich). The amount of lysineformed was determined with an amino acid analyzer fromEppendorf-BioTronik (Hamburg, Germany) by means of ion exchangechromatography and postcolumn derivatization with ninhydrin detection.The glucose content was determined with a sugar analyzer from SkalarAnalytik GmbH (Erkelenz, Germany).

The experimental results are shown in Table 1.

TABLE 1 OD Lysine-HCl Strain Gene (660 nm) g/l DSM5715/pJCl/pEC7lysElysE 9.1 11.1 DSM5715/pJC33/pEC7lysE lysE, lysC 8.7 12.2DSM5715/pJC50/pEC7lysE lysE, lysC, dapA 9.1 12.7 DSM5715/pJC23/pEC7lysElysE, dapA 10.2 13.3 DSM5715/pJC23/ lysE, dapA, dapB 10.9 15.4pEC7dapBlysE

Example 9 Cloning of the aecD Gene into Vector pUC18

Plasmid pSIR21 (Rossol, Thesis, University of Bielefeld 1992) was fullycleaved with the enzymes BglII and EcoRV and the 1.4 kb DNA fragmentcontaining the aecD gene (accession number M89931) (Rossol and Pithier,Journal of Bacteriology 174 (9), 2968-2977 (1992)) from C. glutamicumATCC13032 was isolated. The isolated DNA fragment was ligated to plasmidpUC18 (which had been fully digested with the enzymes BamHI and SmaI)using T4 DNA ligase, as described in Sambrook et al. (Molecular Cloning:A Laboratory Manual (1989), Cold Spring Harbor Laboratory Press). Theligation mixture was transformed to the E. coli strain DH5α. Thetransformants were selected on brain-heart agar plates containing 100mg/l of ampicillin. Plasmid DNA was isolated from one colony. Theplasmid obtained was called pUC18::aecD.

Example 10 Cloning of the dapA Gene into Plasmid pSP72

A dapA gene fragment is isolated from plasmid pJC20 (Cremer, J., Thesis1989, University of Dusseldorf) as an SphI-BamHI fragment. Vector pSP72(Promega Corporation, USA) was fully cleaved with the enzymes SphI andBamHI and treated with alkaline phosphatase. The dapA-carrying fragmentwas ligated to this vector using T4 DNA ligase. The DNA was thentransformed to the E. coli strain XL1 Blue (Bullock, Fernandez andShort, BioTechniques (5), 376-379 (1987)). The transformants wereselected on LB medium containing 100 mg/l of ampicillin. Plasmid DNA wasisolated from one transformant and called pSP72::dapA.

Example 11 Mutagenesis of the dapA Promoter and Preparation of PlasmidspSP72::dapA(MC20) and pSP72::dapA(MA16)

The Quickchange site directed mutagenesis kit from Stratagene was usedfor the mutagenesis of the promoter region. The following primers wereconstructed with the aid of said dapA sequence and used for themutagenesis:

For the preparation of pSP72::dapA (MC20)

Primer dap1 for MC20 (SEQ ID NO: 13)CCA AAT GAG AGA TGG TAA CCT TGA ACT CTA TGA GCA Primer dap2 for MC20SEQ ID NO: 14) GTG CTC ATA GAG TTC AAG GTT ACC ATC TTC CCT CAT TTG G

For the preparation of pSP72::dapA(MA16)

Primer dap3 for MA16 (SEQ ID NO: 15)CCA AAT GAG GGA AGA AGG TAT AAT TGA ACT CTA TGA GCA Primer dap4 for MA16(SEQ ID NO: 16) GTG CTC ATA GAG TTC AAT TAT ACC TTC TTC CCT CAT TTG G

The PCR was carried out as indicated by the manufacturer of theQuickchange site directed mutagenesis kit (Stratagene) using plasmidpSP72::dapA (from Example 10) as the template.

The mutagenesis mixtures were transformed to the E. coli strain XL1Blue. The transformants were selected on LB medium containing 100 mg/lof carbenicillin. Plasmid DNA was isolated from one transformant and theloss of the BstEII cleavage site was controlled by BstEII digestion.Plasmids no longer carrying a BstEII cleavage site exhibited the desiredmutation.

The plasmids obtained were transformed to the dapA-defective E. colimutant RDA8. The transformation mixtures were plated on LB containing100 mg/l of carbenicillin in order to test the complementation of thedapA mutation. DNA was isolated from one transformant in each case andthe plasmids obtained were called pSP72::dapA(MC20) andpSP72::dapA(MA16). The plasmids were sequenced by the chain terminationmethod described in Sanger et al., Proceedings of the National Academyof Sciences of the USA (74), 5463-5467 (1977), using the reverse anduniversal sequencing primers. The sequencing reaction was performed withthe aid of the AutoRead Sequencing Kit (Pharmacia, Freiburg). Theelectrophoretic analysis and detection of the sequencing products werecarried out with the A.L.F. DNA sequencer (Pharmacia, Freiburg).

Example 12 Preparation of plasmids pK19mobsacBaecD::dapA(MC20) andpK19mobsacBaecD::dapA(MA16) (Recloning of the Mutagenized Fragments)

Plasmids pSP72::dapA(MC20) and pSP72::dapA(MA16) (from Example 11) werefully cleaved with the restriction enzymes PvuII and SmaI. The 1450 byPvuII-SmaI fragments carrying the dapA gene with the mutated MC20 orMA16 promoter were ligated to StuI-cleaved vector pUC18::aecD (fromExample 9) using T4 DNA ligase. The ligation mixture was transformed tothe E. coli strain DH5α. The transformants were selected on LB mediumcontaining 100 mg/l of ampicillin. Plasmid DNA was isolated from onetransformant in each case to give plasmids pUC18aecD::dapA(MC20) andpUC18aecD::dapA(MA16).

Plasmids pUC18aecD::dapA(MC20) and pUC18aecD::dapA(MA16) were partiallycleaved with the restriction enzyme EcoRI and fully cleaved with theenzyme SalI to give the 3.0 kb fragment carrying aecD::dapA(MA16) oraecD::dapA(MC20). The fragment was ligated to vector pK19mobsacB (whichhad been cleaved and treated with alkaline phosphatase) (Schäfer et al.,Gene (145), 69-73 (1994)) using T4 DNA ligase. The ligation mixture wastransformed to the E. coli strain DH5 (Hanahan (1985), in: DNA Cloning.A Practical Approach, vol. I, IRL-Press, Oxford, Washington D.C., USA).The transformants were selected on LB medium containing 50 mg/l ofkanamycin. Plasmid DNA was isolated from one transformant in each caseto give plasmids pK19mobsacBaecD::dapA(MC20) andpK19mobsacBaecD::dapA(MA16).

The plasmid DNA was transformed to the E. coli strain S17-1 (Simon,Priefer and Pithier, Bio/Technology (1), 784-791 (1983)). Thetransformants were selected on LB medium containing 50 mg/l ofkanamycin. Plasmid DNA was isolated from one transformant in each caseand checked. The strains obtained were calledS17-1/pK19mobsacBaecD::dapA(MC20) and S17-1/pK19mobsacBaecD::dapA(MA16).

Example 13 Preparation of the C. glutamicum StrainsDSM5715aecD::dapA(MC20) and DSM5715aecD::dapA(MA16)

Plasmids pK19mobsacBaecD::dapA(MC20) and pK19mobsacBaecD::dapA(MA16)were transferred from S17-1/pK19mobsacBaecD::dapA(MC20) andS17-1/pK19mobsacBaecD::dapA(MA16) (from Example 12) to the C. glutamicumstrain DSM5715 by the conjugation method (Schäfer et al., Journal ofBacteriology (172), 1663-1666 (1990)). For selection of thetransconjugants, the conjugation mixtures were plated on brain-heartmedium containing nalidixic acid and kanamycin. The transconjugantsobtained were incubated overnight in 10 ml of brain-heart medium.Aliquots were then plated on plates containing sucrose (brain-heart agarcontaining 10% of sucrose) in order to select for loss of sucrosesensitivity. Sucrose-resistant clones were isolated and checked again onagar plates containing chloramphenicol and kanamycin (brain-heart mediumcontaining 15 mg/l of kanamycin and brain-heart medium containing 10mg/l of chloramphenicol).

Colonies exhibiting the following phenotype were isolated:

sucrose resistantkanamycin sensitivechloramphenicol sensitive

The insertion of the dapA gene fragment into the aecD gene was checkedby the Southern blot method (Sambrook et al., Molecular Cloning: ALaboratory Manual (1989), Cold Spring Harbor Laboratory Press).

Example 14 Preparation of the C. glutamicum StrainsDSM5715aecD::dapA(MC20)/pEC7lysE, DSM5715aecD::dapA(MA16)/pEC7lysE,DSM5715aecD::dapA(MC20)/pEC7, DSM5715aecD::dapA(MA16)/pEC7 andDSM5715/pEC7

As described in Example 3, the lysE gene is present in vector pEC7. Thisplasmid pEC7lysE and plasmid pEC7 were introduced into the strainsDSM5715aecD::dapA(MC20), DSM5715aecD::dapA(MA16) and DSM5715 (fromExample 13) by means of electroporation (Haynes 1989, FEMS MicrobiologyLetters 61, 329-334) to give C. glutamicumDSM5715aecD::dapA(MC20)/pEC7lysE, DSM5715aecD::dapA(MA16)/pEC7lysE,DSM5715aecD::dapA(MC20)/pEC7, DSM5715aecD::dapA(MA16)/pEC7 andDSM5715/pEC7. The transformants were selected on brain-heart agarcontaining 25 mg/l of kanamycin. Plasmid DNA was isolated from onetransformant in each case and checked.

The following strains were obtained in this way:

DSM5715aecD::dapA(MC20)/pEC7lysE,DSM5715aecD::dapA(MA16)/pEC7lysE,DSM5715aecD::dapA(MC20)/pEC7,DSM5715aecD::dapA(MA16)/pEC7, andDSM5715/pEC7.

Example 15 Preparation of Lysine with the Strains Prepared in Example 14

After precultivation in CgIII medium (Kase & Nakayama, Agricultural andBiological Chemistry 36 (9), 1611-1621 (1972)), the strainsDSM5715aecD::dapA(MC20)/pEC7lysE, DSM5715aecD::dapA(MA16)/pEC7lysE,DSM5715aecD::dapA(MC20)/pEC7, DSM5715aecD::dapA(MA16)/pEC7 andDSM5715/pEC7 were cultivated in MM production medium as described inExample 8. After incubation for 48 hours, the optical density at 660 nmand the concentration of L-lysine formed were determined.

The experimental results are shown in Table 2.

TABLE 2 OD Lysine•HCl Strain (660 nm) g/lDSM5715aecD::dapA(MC20)/pEC7lysE 13.3 13.8DSM5715aecD::dapA(MA16)/pEC7lysE 12.4 14.3 DSM5715/pEC7 13.3 11.5DSM5715aecD::dapA(MA16)/pEC7 13.3 12.9 DSM5715aecD::dapA(MC20)/pEC7 14.012.8

1. L-Lysine-producing corynebacteria with an enhanced lysE gene, in which additionally genes selected from the group consisting of the dapA gene, the lysC gene, the pyc gene and the dapB gene, individually or together, are enhanced.
 2. Corynebacteria as claimed in claim 1 in which the dapB gene is enhanced.
 3. Corynebacteria as claimed in claim 1 in which the dapB gene, which additionally contains the 5′ end upstream from the translation start of this gene, said 5′ end being shown in SEQ ID No. 1, is enhanced.
 4. Corynebacteria as claimed in claim 1 which contain the MC20 or MA16 mutation of the dapA promoter shown respectively in SEQ ID No. 5 and SEQ ID No.
 6. 5. Isolated DNA originating from Corynebacterium and capable of replication in corynebacteria, which contains at least the nucleotide sequence coding for the 5′ end upstream from the translation region of the dapB gene, shown in SEQ ID No.
 1. 6. DNA capable of replication, as claimed in claim 5, comprising the nucleotide sequence shown in SEQ ID No.
 1. 7. Corynebacteria as claimed in claim 1 in which the dapA gene and the lysC gene are enhanced.
 8. Corynebacteria as claimed in claim 1 wherein the genes are enhanced through over-expression.
 9. Corynebacteria as claimed in claim 2 wherein the dapB gene is over-expressed.
 10. Corynebacteria as claimed in claim 3 wherein the dapB gene is over-expressed.
 11. The DNA of claim 5 which is recombinant.
 12. Escherichia coli K-12 strain DH5α/pEC7dapBlysE, deposited as DSM12875.
 13. Corynebacterium glutamicum strain DSM5715aecD::dapA(MA16), deposited as DSM12867.
 14. Corynebacterium glutamicum strain DSM5715aecD::dapA(MC20), deposited as DSM12868. 